Higher vertebrates can synthesize a vast number of different antibody molecules. This diversity is generated by combinations of many different immunoglobulin heavy and light chains. The genes for the large number of chains are created during ontogeny from multicopy gene segments as organized in the germ line. The large combinatorial diversity of the genes is augmented by the diversity of variations in the precise points of juncture. Somatic mutations, probably point mutations, also contribute to the diversity of antibodies. In view of our discovery of hypermutation in a mouse pre-B-lymphocyte line, it seems very likely that somatic mutations are not only selected from among those that arise at the normal spontaneous rate, but that there is a mutator that causes hypermutation at some period during the ontogeny of B lymphocytes. The aim of the proposed study is to define the mutator, i.e., to answer the questions of where, how, and when the mutator works. This will be attempted by conducting three types of experiments: (i) measuring mutation rates at the variable and constant region gene segments of the immunoglobulin loci and at two nonimmunoglobin loci in cell lines of various different stages; (ii) isolating some of the mutants and sequencing the relevant DNA segments; (iii) introducing into various cell lines DNA sequences to be mutated and then recovered for analysis. This will be done by transfection of shuttle vectors, which replicate in both mammalian cells and bacteria, and by infection of the viruses with DNA insertions of interest.